Catalases [hydrogen peroxide: hydrogen peroxide oxidoreductases (EC 1.11.1.6)] are enzymes which catalyze the conversion of hydrogen peroxide (H.sub.2 O.sub.2) to oxygen (O.sub.2) and water (H.sub.2 O) according to the following formula: ##STR1##
These ubiquitous enzymes have been purified from a variety of animal tissues, plants and microorganisms (Chance and Maehly 1955 Methods Enzymol. 2: 764-791; Jones and Wilson 1978 in H. Sigel (ed.), Metal Ions in Biological Systems, Vol. 7, Marcel Dekker Inc., New York).
Nearly all forms of the enzyme which have been characterized consist of four polypeptide subunits, each having a molecular weight of 50,000 to 60,000 and containing one protohemin prosthetic group per subunit (Wasserman and Hultin 1981 Arch. Biochem. Biophys. 212: 385-392; Hartig and Ruis 1986 Eur. J. Biochem. 160: 487-490). Bovine liver catalase has been the most extensively studied variety of this enzyme [Schonbaum and Chance 1976 in The Enzymes (P. D. Boyer, ed.) 3rd edn., vol. 13, pp. 363-408, Academic Press, New York]. The complete amino acid sequence and three dimensional structure of bovine liver catalase are known (Schroeder, et al., 1982 Arch. Biochem. Biophys. 214: 397-412; Murthy, et al., 1981 J. Mol. Biol. 152: 465-499).
Although less well-studied from a biochemical and biophysical standpoint, catalases from filamentous fungi have several characteristics that distinguish them from their mammalian counterparts. While similar in subunit number and heme content, fungal catalases are substantially larger molecules than those from other organisms, having subunit molecular weights ranging from 80,000 to 97,000 (Vainshtein, et al., 1986 J. Mol. Biol. 188: 63-72; Jacob and Orme-Johnson 1979 Biochem. 18: 2967-2975; Jones, et al., 1987 Biochim. Biophys. Acta 913: 395-398). More importantly, catalases from fungi such as Aspergillus niger are more stable than beef liver catalase to proteolysis and to inactivation by glutaraldehyde, SDS, and have lower affinity for catalase inhibitors such as cyanide, azide and fluoride (Wasserman and Hultin 1981 Arch. Biochem. Biophys. 212: 385-392). In addition, A. niger catalase is significantly more stable than beef liver catalase when subjected to extremes of pH, hydrogen peroxide, and temperature (Scott and Hammer 1960 Enzymologia 22: 229- 237). Although fungal catalases offer stability advantages, the corresponding mammalian enzymes such as beef liver catalase appear to have higher catalytic activity (Gruft, et al., 1978; Kikuchi-Torii, et al., 1982). However, since enzyme stability is an important factor in the biotechnological utilization of enzymes, there has been considerable interest in the use of fungal catalases, especially for applications involving neutralization of high concentrations of hydrogen peroxide. Vasudevan and Weiland (1990 Biotechnol. Bioeng. 36: 783-789) observed that the rate of deactivation in H.sub.2 O.sub.2 was at least an order of magnatude lower for A. niger catalase than for beef liver catalase. The differences in stability of these two enzymes can probably be attributed to differences in structural characteristics and composition of the proteins [Vasudevan and Weiland 1990 Biotechnol. Bioeng. 36: 783-789].
Catalase preparations from A. niger are sold commercially for diagnostic enzyme kits, for the enzymatic production of sodium gluconate from glucose, for the neutralization of H.sub.2 O.sub.2 waste, and for the removal of H.sub.2 O.sub.2 and/or generation of O.sub.2 in foods and beverages. Traditionally, beef liver catalase has been the preferred enzyme for diagnostic purposes and for pharmaceutical-related applications (e.g., contact-lens cleaning/disinfection/H.sub.2 O.sub.2 neutralization). However, recent outbreaks of a slow-virus disease known as BSE (bovine spongiform encephalopathy) in European cattle herds and fear that this disease might be spread to man [Dealler and Lacey 1991 Nutr. Health (Bicester) 7: 117-134; Dealler and Lacey 1990 Food Microbiol. 7: 253-280] have aroused interests in finding alternatives to beef liver catalase for most industrial applications. Little information has been published regarding the regulation of catalase synthesis in A. niger. However, it has been observed that catalase is produced in response to the generation of H.sub.2 O.sub.2 during growth of the organism on glucose or fatty acids. For example, during the metabolism of glucose, H.sub.2 O.sub.2 is formed by oxidation of the sugar to give gluconate. This reaction is catalyzed by the enzyme glucose oxidase: ##STR2##
Cellular metabolism of fatty acids, which occurs in specialized organelles known as peroxisomes, also yields H.sub.2 O.sub.2 which induces the formation of catalase. However, in a distantly related fungus (yeast), Saccharomyces cervisiae, a specific catalase is induced during growth on fatty acids. This catalase, termed catalase-A (atypical), is localized chiefly in peroxisomes where fatty acid oxidation occurs. A second S. cerevisiae enzyme, catalase-T (typical) is a soluble cytoplasmic enzyme which is synthesized in response to a variety of other metabolic and environmental stresses. These two yeast catalases are the products of two different nuclear genes, designated CTA1 and CTT1. Similarly, two catalase genes have been isolated from A. niger (Genencor International, Inc., unpublished). The A. niger catA gene, cloned by cross-hybridization to the yeast CTA1 gene, encodes a catalase enzyme which is induced primarily during growth on fatty acids and is presumably peroxisomal. This enzyme (catalase-A) is not of commercial importance at this time, however, a second cloned A. niger catalase gene, designated as catR, encodes a soluble cytoplasmic enzyme (catalase-R) which represents the major activity in commercial catalase preparations.
Because of the obvious commercial interest in A. niger catalases, it would be desirable to obtain A. niger strains which produce increased levels of the catR gene product. Furthermore, it would be a significant advantage to effect high levels of catalase synthesis without the need to generate hydrogen peroxide as an inducer. Concomitant with the generation of hydrogen peroxide is the formation of sodium gluconate which represents a waste disposal problem. Thus, it is also highly desirable to minimize the production of gluconate in large scale fermentations with catalase production strains of A. niger. This invention discloses a solution for simultaneously accomplishing all of these objectives.